X-ray fluorescence microscopes offer the highest sensitivity for studies of the role of trace metals in cells, and they provide essential informatio for understanding the ultrastructural targeting of nanoparticles used for potential cancer therapies. With ARRA support from the NIH, co-PI Woloschak et al. have obtained the Bionanoprobe at Argonne's Advanced Photon Source (the nation's premier hard x-ray synchrotron light source facility). This is a first-of-its-kind instrument for x-ray fluorescence studies of trace elements in cells and tissue sections which offers a thousandfold improvement in sensitivity compared to electron microprobes, cryo transfer conditions to work with fully hydrated specimens at the highest preparation fidelity possible, and 3D tomographic imaging capabilities. Our proposal is to devote the scientific and technical effort needed to realize this new instrument's potential. Since hard x-ray microscopes are able to image samples with thicknesses ranging up to tens of micrometers, we can study whole cells and tissue sections in 3D in a way that electron microscopes cannot, and with a combination of sub-50 nm spatial resolution and sensitive trace element quantitation available in no other method. Cryogenic conditions are required to minimize radiation damage effects and maximize the fidelity of ultrastructure and trace element concentrations, so we need to develop and validate the novel cryogenic sample preparation methods appropriate for our unique sample types. X-ray fluorescence is relatively blind to the light elements (such as organic materials and water) that comprise nearly all the mass and ultrastructure of cells. Fortunately, we have pioneered several methods that can make visible what was once dark, and we will develop a variant of these approaches (ptychography, a method of scanned coherent imaging that delivers quantitative phase contrast and spatial resolution beyond the limit of x-ray optics) and turn this into a routin, simultaneous-with-fluorescence imaging method for users of the Bionanoprobe. To validate these approaches and work from the beginning on a crucial biomedical research project, we will do this in the context of ongoing research in the use of DNA-conjugated nanoparticles containing titanium and/or gadolinium that are meant to target mitochondria for the treatment and imaging of prostrate, breast, and other cancers. In this way, we will develop the methods needed to fully realize the investment NIH has already made in the Bionanoprobe.